Discovery of regulatory t cells programmed to suppress an immune response

ABSTRACT

A method to treat an autoimmune disease is provided. The method involves administration of interleukin-15 receptor (IL-15R) agonists in an amount effective to ameliorate a symptom of the autoimmune disease. The invention also involves a method to treat an autoimmune disease by ex-vivo expansion of CD44+CD122+Kir+ CD8+ Treg cells and administration of the CD44+CD122+Kir+ CD8+ Treg cells. Compositions comprising CD44+CD122+Kir+ CD8+ Treg cells are also provided. Methods for stimulating an immune response to an antigen are also provided.

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) of U.S. provisional application Ser. No. 61/405,696, filed Oct. 22, 2010, the content of which is incorporated by reference herein in its entirety.

FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under NIH Grant AI 037562. Accordingly, the Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

Achieving a balance between induction of protective immunity against pathogens and maintenance of self-tolerance is a central feature of the adaptive immune system. Although negative selection in the thymus removes the majority of clones that express T cell receptors (TCR) with high affinity for self-peptide MHC products, this process is incomplete. A significant fraction of mature peripheral T cells that respond to self-peptide-MHC complexes may differentiate into effector cells in the context of inflammatory stimuli (Bouneaud et al., 2000; Goldrath and Bevan, 1999; Slifka et al., 2003). Although this process is constrained by abortive or defective TCR signaling resulting in cellular elimination (AICD) or inactivation (Martin et al., 1999; Kearney et al., 1994), these cell-intrinsic mechanisms may not suffice to prevent the development of autoimmune disorders (Anderton et al., 2001; Panoutsakopoulou et al., 2001). There is increasing evidence that self tolerance may also depend on inhibitory interactions between effector T cells and regulatory or suppressive cells (Littman and Rudensky, 2010). However, regulatory T cells that are genetically programmed to inhibit development of autoantibody formation and autoimmune diseases have not been defined. Due to the severity and breath of autoimmune diseases such as lupus and rheumatoid arthritis, there is a great need for effective treatments of such diseases.

SUMMARY OF THE INVENTION

The invention, relates in some aspects, to the discovery of a subpopulation of CD8⁺ regulatory T cells that are essential for maintenance of self tolerance and prevention of autoimmune disease. In some aspects, the invention involves a method for treating an autoimmune disease. The method involves administering to a subject in need of such treatment an interleukin-15 receptor (IL-15R) agonist in an amount effective to ameliorate a symptom of the autoimmune disease. In some embodiments, the autoimmune disease is selected from the group consisting of systemic lupus erythematosus, chronic graft versus host disease, rheumatoid arthritis, insulin-dependent diabetes mellitus, multiple sclerosis, psoriasis, inflammatory bowel disease, Sjogren's syndrome, Graves disease, Crohn's disease, Waldenstrom's macroglobulinemia, hyperviscosity syndrome, monoclonal gammopathy of undetermined origin, POEMS syndrome, myeloma, macroglobulinemia, and cold agglutinin disease. In some embodiments, the autoimmune disease comprises antibodies to a self antigen and the subject has the antibodies to the self antigen. In some embodiments, the IL-15R agonist is a IL-15 polypeptide that comprises a sequence at least 90% identical to the amino acid sequence of SEQ ID NO:1 and binds an IL-15R. In some embodiments, the agonist is a IL-15 polypeptide that comprises a sequence at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO:1. In some embodiments, the agonist is a IL-15 polypeptide that comprises SEQ ID NO:1. In some embodiments, the agonist is an anti-IL15R antibody or an antigen-binding fragment thereof. In other embodiments, the agonist is a heterocomplex of IL-15 linked to soluble IL-15 receptor alpha chain via a GS linker.

According to one aspect of the invention, a method for treating an autoimmune disease in a subject using CD44⁺CD122⁺Kir⁺ CD8⁺ Treg cells is provided. The method comprises isolating T cells from the subject in need of such treatment, wherein the isolated T cells comprise a number of CD44⁺CD122⁺Kir⁺ CD8⁺ Treg cells; growing the isolated cells in a culture medium containing IL-15 and IL-21 in the presence of Concanavalin A activated and irradiated syngeneic CD4 T cells until the number of CD44⁺CD122⁺Kir⁺ CD8⁺ Treg cells increases by at least 5%, thereby producing a population of cells enriched with CD44⁺CD122⁺Kir⁺ CD8⁺ Treg cells; and administering CD44⁺CD122⁺Kir⁺ CD8⁺ Treg cells from the population of cells to the subject in an amount effective to ameliorate a symptom of the autoimmune disease. The CD44⁺CD122⁺Kir⁺ CD8⁺ Treg cells from the population of cells are separated from the culture medium before administration to the subject. In some embodiments, the CD8⁺ T Cells are isolated from the subject. The CD44⁺CD122⁺Kir⁺ CD8⁺ Treg cells from the population of cells may be administered by intravenous injection. In some embodiments, the isolated cells are grown until the number of CD44⁺CD122⁺Kir⁺ CD8⁺ Treg cells increases by at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, at least 90%, at least 95% or at least 100%. The population of cells enriched with CD44⁺CD122⁺Kir⁺ CD8⁺ Treg cells is further enriched by depleting a population of non-CD44⁺CD122⁺Kir⁺ CD8⁺ Treg cells. In some embodiments, a portion of CD44⁺CD122⁺Kir⁺ CD8⁺ Treg cells from the population of cells is administered to the subject. In some embodiments, all CD44⁺CD122⁺Kir⁺ CD8⁺ Treg cells from the population of cells are administered to the subject.

According to some aspects of the invention, a composition comprising CD8⁺ cells, wherein at least 5% of the CD8⁺ cells in the composition are CD44⁺CD122⁺Kir⁺ CD8⁺ Treg cells is provided. In some embodiments, at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, at least 90%, at least 95% or 100% of the CD8⁺ cells in the composition are CD44⁺CD122⁺Kir⁺ CD8⁺ Treg cells. The invention also involves, in some aspects, a pharmaceutical composition comprising a composition of CD8⁺ cells, wherein at least 5% of the CD8⁺ cells in the composition are CD44⁺CD122⁺Kir⁺ CD8⁺ Treg cells, and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition further comprises an immunosuppressive agent.

According to some aspects of the invention, a method for stimulating an immune response to an antigen is provided. The method comprises administering to a subject having the antigen and in need of immune stimulation to the antigen an agent that inhibits the proliferation and/or activity of CD44⁺CD122⁺Kir⁺ CD8⁺ Treg cells in an amount effective to stimulate an immune response in the subject to the antigen. In some embodiments, the subject has an infection with a pathogen and the pathogen comprises the antigen. In some embodiments, the subject has cancer, the cancer comprises the antigen, and the antigen is a tumor antigen. The antigen may be administered to the subject in a vaccine. The agent that inhibits the proliferation and/or activity of CD44⁺CD122⁺Kir⁺ CD8⁺ Treg cells can be an anti IL-15 antibody, an antibody that binds CD44⁺CD122⁺Kir⁺ CD8⁺ Treg cells, an anti-CD8 antibody, or an anti-Kir antibody. In some embodiments, the antibody is a bispecific antibody comprising a first antigen-binding domain that binds to a first surface marker present on the CD44⁺CD122⁺Kir⁺ CD8⁺ Treg cells; and a second antigen-binding domain that binds to a second surface marker present on the CD44⁺CD122⁺Kir⁺ CD8⁺ Treg cells. The first and second surface markers on the CD44⁺CD122⁺Kir⁺ CD8⁺ Treg cells may be selected from the group consisting of CD44, CD122, Kir, and CD8. In some embodiments, the agent is a composition comprising a sub-optimal dose of a first antibody that binds a first surface antigen present on CD44⁺CD122⁺Kir⁺ CD8⁺ Treg cells, and a sub-optimal dose of a second antibody that binds a second surface antigen present on the CD44⁺CD122⁺Kir⁺ CD8⁺ Treg cells, wherein the first surface antigen is different from the second surface antigen. The first antibody binds an antigen selected from the group consisting of CD44, CD122, Kir, and CD8 and the second antibody binds an antigen selected from the group consisting of CD44, CD122, Kir, and CD8.

Each of the embodiments and aspects of the invention can be practiced independently or combined. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including”, “comprising”, or “having”, “containing”, “involving”, and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

These and other aspects of the inventions, as well as various advantages and utilities will be apparent with reference to the Detailed Description. Each aspect of the invention can encompass various embodiments as will be understood. All documents identified in this application are incorporated in their entirety herein by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows Qa-1 dependent suppression of Ab response by CD44⁺CD122⁺ CD8 T cells. FIG. 1A shows the percentage and number of CD44⁺CD122⁺ CD8 T cells from naïve and KLH/CFA immunized (d 8) WT or IL-15^(−/−) mice. FACS profiles are shown after gating on CD3⁺CD8⁺ cells. FIG. 1B shows Ab response after transfer of B, CD4 and CD8 cells into Rag2^(−/−) mice. 2×10⁶ WT naïve B cells were transferred along with 1×10⁶ CD25⁺ depleted CD4 cells from B6.Qa-1(WT) or B6.Qa-1(D227K) mice into Rag2^(−/−) hosts. CD44⁺CD122⁺or CD44⁺CD122⁻ CD8⁺ T cells isolated from KLH/CFA immunized WT B6 mice were also transferred into these Rag2^(−/−) hosts before recipients were immunized i.p. with 100 μg NP19-KLH in CFA. At day 10, mice were challenged i.p. with 50 μg NP19-KLH in IFA and NP specific Ab responses were measured by ELISA seven days later. FIG. 1C shows defective inhibition by CD8 Treg leads to autoantibody generation. Anti-thyroglobulin and anti-insulin Ab generated in Rag2 recipients were measured by ELISA at day 50.

FIG. 2 shows that CD44⁺CD122⁺Ly49⁺ CD8 T cells account for Qa-1 restricted suppressive activity. FIG. 2A: WT B6 mice were immunized i.p. with 100 μg KLH in CFA and 10 days later surface expression of CD44, CD122, Ly49 subtypes, CD8 and CD62L was analyzed. Numbers are percentage of cells expressing each surface protein. FIG. 2B shows the Ab response after transfer of B, CD4 and CD8 cells into Rag2^(−/−) hosts. 2×10⁶ WT naïve B cells were transferred along with 0.5×10⁶ CD25⁺ depleted CD4 cells from B6.Qa-1(WT) or B6.Qa-1(D227K) mice into Rag2^(−/−) hosts. CD44⁺CD122⁺Ly49⁺ CD8⁺ T cells were isolated from KLH/CFA immunized WT B6 mice and 0.15×10⁶ cells were transferred into Rag2^(−/−) hosts. Immediately after cell transfer, Rag2^(−/−) recipients were immunized i.p. with 100 μg NP19-KLH in CFA. At day 10, mice were challenged i.p. with 50 μg NP19-KLH in IFA and high affinity NP specific Ab responses were measured by ELISA seven days after challenge.

FIG. 3 shows the expansion of TFH and GC B cells in B6-Yaa mice. Spleen cells from age matched (2 mo and 8 mo) WT B6 and B6-Yaa mice were stained with CD4, ICOS and CD200 antibodies for TFH cells and B220, IgM and Fas antibodies for GC B cells. The absolute numbers of TFH and GC B cells are shown in the right panel.

FIG. 4 shows the impaired suppression of Ab response by CD8⁺ T cells from B6-Yaa mice. WT naïve B cells were transferred along with CD25⁺depleted CD4 T cells from Qa-1 (WT) or Qa-1 (D227K) mice into Rag2^(−/−) hosts. FACS sorted CD44⁺CD122⁺ CD8 T cells isolated from KLH/CFA immunized WT or B6-Yaa mice were transferred into Rag2^(−/−) hosts. Immediately after cell transfer, Rag2^(−/−) recipients were immunized i.p. with 100 μg NP19-KLH in CFA and reimmunized i.p. with 50 μg NP19-KLH in IFA 20 days later.

FIG. 5 shows the suppressive activity by CD44⁺ICOSL⁺ CD8 T cells. Ab response after transfer of B, CD4 and CD8 cells into Rag2^(−/−) hosts. 2×10⁶ WT naïve B cells were transferred along with 0.5×10⁶ CD25⁺ depleted CD4 cells from B6.Qa-1(WT) or B6.Qa-1(D227K) mice into Rag2^(−/−) hosts. CD44⁺ICOSL⁺ CD8⁺ T cells were sorted by FACS from spleen cells of KLH/CFA immunized WT B6 mice. 0.15×10⁶ sorted CD44⁺ICOSL⁺ CD8 cells were transferred into Rag2^(−/−) hosts. Immediately after cell transfer, Rag2^(−/−) recipients were immunized i.p. with 100 μg NP19-KLH in CFA. At day 10, mice were challenged i.p. with 50 μg NP19-KLH in IFA and high affinity NP specific Ab responses were measured by ELISA seven days after challenge.

FIG. 6 shows the amino acid sequence of human IL-15 (SEQ ID NO: 1; NCBI Reference Sequence: NP_(—)000576.1).

FIG. 7 shows that vaccination with 2×10⁵ GM-CSF-irradiated (3500 rads) B16 cells confers increased survival after B16 tumor inoculation in Qa-1(D227K) knock in compared with WT B6 mice.

FIG. 8 shows the genetic characterization of CD44⁺CD122⁺Ly49⁺ CD8⁺ Treg cells. FIG. 8A shows a comparison of the gene expression profile between Ly49⁺ and Ly49⁻ CD44⁺CD122⁺ CD8 T cells by gene microarray analysis. B6 WT mice were immunized i.p. with KLH in CFA and Ly49⁺ and Ly49⁻ CD8 T cells were purified from spleen 7 days post immunization. Gene expression was analyzed using an Affymetrix microarray system and differential gene expression was assessed by Multiplot analysis program. 125 genes were upregulated and 42 genes were downregulated in Ly49⁺ cells compared to Ly49⁻ cells. FIG. 8B shows the validation of Helios expression by RT-PCR: 5.3 fold increase of Helios expression was observed in Ly49⁺ CD8⁺ T cells in gene microarray analysis. Independent cDNA prepared from Ly49⁺ and Ly49⁻ CD8 T cells were used to validate the increase of Helios expression in Ly49⁺ CD8 T cells by RT-PCR. FIG. 8C shows that innate receptors are highly upregulated in Ly49⁺ CD8⁺ T cells: Gene expression profiling of Ly49⁺ CD8⁺ T cells in comparison to memory type Ly49⁻ CD8 T cells revealed that receptors normally expressed in NK cells are prominently upregulated in Ly49⁺ T cells at mRNA level.

FIG. 9 shows the phenotypic characterization of Helios⁺Ly49⁺ CD8⁺ T cells. In FIG. 9A, FACS analysis of Ly49⁺ CD8 T cells revealed that Helios is expressed in 50% of Ly49⁺ CD8 T cells. Helios⁺ Ly49⁺ CD8 T cells display also higher level of integrin expression (VLA-2 and VLA-4) that may have functional significance in the cell contact dependent recognition of target cells. FIG. 9B shows the requirement of IL-15 for the development of Helios⁺Ly49⁺ CD8 T cells: IL-15 deficient mice show a dramatic reduction in Ly49⁺ CD8 T cells (18.9% of CD44⁺Cd122⁺ CD8 T cells in WT vs. 3% of CD44⁺CD12⁺ CD8 T cells in IL-15^(−/−) mice). Ly49⁺ CD8⁺ T cells from Il-15^(−/−) mice do not express Helios suggesting that the development of Helios⁺ Ly49⁺ CD8⁺ T cells is IL-15 dependent. FIG. 9C shows the correlation between IL-15 responsiveness and level of Ly49 expression: Helios⁺ Ly49⁺ CD8 T cells may belong to the most IL-15 sensitive CD8 T cells. Helios⁺ Ly49⁺ CD8 T cells express higher level of CD122 and Ly49 compared to Helios⁻ Ly49⁺ CD8 T cell counterpart. In vitro stimulation of Ly49⁺ CD8 T cells shows that Ly49⁺ expression is tightly associated with the strength of IL-15 stimulation. The extreme responsiveness of Helios⁺ Ly49⁺ CD8 T cells can be exploited in the in vitro expansion of CD8⁺ Treg using minute amount of IL-15 in the cell culture. FIG. 9D shows that Helios deficient mice lack Ly49^(hi) CD8 T cells: we have generated bone marrow (BM) chimera in which Rag2^(−/−) mice were reconstituted with Helios WT or Helios KO BM cells. BM chimera generated with WT BM cells develop CD44⁺ CD122⁺ and CD44⁺CD122⁺Ly49⁺ CD8 T cells that are similar to WT B6 mice in their frequency. Helios KO BM chimera, however, show an increased CD44⁺CD122⁺ CD8 T cells and a dramatic reduction of Ly49⁺ CD8 T cells. FACS analysis for Helios expression confirms the Helios KO genotype. FIG. 9E shows the increase of activated CD4 and CD8 T cells in recipients of Helios KO BM cells: FACS analysis of spleen cells from Helios WT and KO BM revealed that Helios KO BM chimera have approximately 2 fold increase in activated CD4⁺ T cells (CD4⁺CD44⁺CD62L^(lo)) and 6 fold increase in activated CD8⁺ T cells (CD8⁺CD44⁺CD62L^(lo)). FIG. 9F shows the impaired suppressive activity of Helios deficient CD8⁺ T cells: Rag2^(−/−) mice were transferred with B cells from WT B6 mice, CD25 depleted CD4⁺ T cells from WT B6 mice and Ly49⁺ CD8 T cells purified from Helios WT and KO BM chimera. Helios WT and KO BM chimera were immunized i.p. with KLH in CFA 7 days prior to CD8 preparation. Rag2^(−/−) hosts were immunized i.p. with NP19-KLH/CFA immediately after cell transfer. While Rag2^(−/−) hosts that received WT Ly49⁺ CD8 T cells show an efficient suppression of anti-NP Ab response, Helios KO Ly49⁺ CD8 T cell recipients show a similar level of anti-NP Ab response to Rag2^(−/−) mice that did not receive CD8 T cells.

FIG. 10 shows CD8 Treg-mediated immunotherapy in autoimmune disease: CIA model. FIG. 10A shows the prevention of CIA by infusion of CD8⁺ Treg: WT B and CD25 depleted CD4 cells were sorted from spleen cells of WT B6 mice. Ly49⁺ and Ly49⁻ CD8 T cells are sorted from spleen and lymph nodes. Collagen type II cells were transferred into Rag2^(−/−) Prfl^(−/−) mice followed by arthritis induction at d0 and boosted at d21. CD8 Treg were infused at d21 and d35 additionally. CIA development was monitored. Sorted CD44⁺CD122⁺Ly49⁺ or Ly49⁻ CD8 cells were cultured in the presence of 2 ng/ml IL-15/IL-15Rα complex for 1 week. Rag2^(−/−)Prfl^(−/−) mice were transferred with B, CD4 and in vitro expanded CD8 T cells (d0, 21 and 35) and CIA development monitored in FIG. 10B. FIG. 10C shows a therapeutic application of CD8 Treg: 1.5×10⁵ in vitro expanded Ly49⁺ CD8 T cells were transferred into B6 mice that developed arthritis in an average score of 3.5 at d 0 and d18. 0.75 mg/kg methotrexate (MTX) was given from d5 to d17 every two days.

DETAILED DESCRIPTION OF THE INVENTION

The present invention, in one aspect, relates to the discovery of a subpopulation of CD8⁺ regulatory T cells that are essential for maintenance of self tolerance and prevention of autoimmune disease. These CD8 T cells are programmed to suppress rather than activate immunity and represent an essential regulatory element of the immune response and a guarantor of self tolerance.

The specialized regulatory CD8⁺ T cells selectively suppress CD4⁺ follicular helper T cell (TFH) activity through recognition of class I MHC peptide Qa-1 (mouse homolog of human leukocyte antigen E (HLA-E)) expressed at the surface of TFH cells and dampen autoantibody responses. An analysis of the surface phenotype of these CD8⁺ regulatory (Treg) cells indicated that they expressed high levels of CD44 and CD122, relatively lower levels of CXCR5 and ICOSL, and also express the Kir receptor (immunoglobulin like killer inhibitory receptor; designated Ly49 in mice). DNA microarray with cDNA prepared from mouse Ly49⁺ and Ly49⁻ CD8⁺ T cells also identified the transcription factor Helios as a potential master transcription factor for genes that are important for the CD8 Treg development and function.

Helios is a member of the Ikaros family of zinc finger regulators. Helios is highly expressed at early stages of T cell development and also expressed in a subset (˜70%) of FoxP3⁺CD4⁺ Treg cells; however, Helios expression is not dependent on FoxP3 expression. Mature B cells, dendritic cells and myeloid cells do not express Helios. Helios expression and function in the CD8 T cell pool has not been reported. The inventors have shown that Helios is exclusively expressed by Ly49⁺ CD8 Treg within the CD8 T cell pool and expression of this transcription factor in Ly49⁺ T cells is associated with their suppressive function. The presence of Helios protein and mRNA can detected by intracellular staining of Helios using antibodies and quantitative PCR (Expression of Helios, an Ikaros Transcription Factor Family Member, Differentiates Thymic-Derived from Peripherally Induced Foxp3⁺ T Regulatory Cells; Thornton et al. J Immunol 2010 184:3433-3441). Helios expression in cells can be detected, for example, by testing an aliquot of the cells for the presence of Helios protein or mRNA. Expression of Helios protein can be detected by staining spleen cells from mice (or PBMC from humans) with surface markers, for example CD3, CD8, CD44, CD122 and Ly49 (KIR in humans). Cells are then fixed with fixation buffer (4% paraformaldehyde in phosphate-buffered saline) and washed with permeabilization buffer (0.1% saponin and 0.009% sodium azide in phosphate-buffered saline). Subsequent staining with anti-Helios antibody in permeabilization buffer allows detection of the expression (as a %) of Helios in this subset of CD8⁺ T cells and allows definition of additional surface markers expressed by Helios⁺ CD8 cells in mouse and man.

Expression of CD122 on their surface is in accord with their dependence on IL-15 for both development and function. Expression of the class I MHC receptor Ly49 by CD8 cells has also been associated with enhanced responsiveness to IL-15 (Coles et al., 2000; Judge et al., 2002; Anfossi et al., 2004). The CD44⁺CD122⁺Kir⁺Helios⁺ CD8⁺ T cells, which represent 3-5% of CD8 T cells, account for virtually all of the Qa-1-restricted suppressive activity in this T cell subset.

The specialized regulatory CD8⁺ T cells are used according to the invention to treat autoimmune diseases. As used here, the specialized regulatory CD8⁺ T cells include CD44⁺CD122⁺Helios⁺ CD8⁺ Treg cells, CD44⁺CD122⁺Helios⁺ Kir⁺CD8⁺ Treg cells, and/or CD44⁺CD122⁺Kir⁺ CD8⁺ Treg cells.

Aspects of the invention involve methods of treating autoimmune diseases by isolating T cells from the subject in need of such treatment, growing the cells in a culture medium enriched with IL-15 and IL-21 in the presence of Concanavalin A activated and irradiated syngeneic CD4 T cells such that the population of CD44⁺CD122⁺Helios⁺ CD8⁺ Treg cells increases in number by at least 5%, thereby producing a population of cells enriched with CD44⁺CD122⁺Helios⁺ CD8⁺ Treg cells. Then CD44⁺CD122⁺Helios⁺ CD8⁺ Treg cells from the population are administered to the subject in an amount effective to ameliorate a symptom of the autoimmune disease. In some embodiments, CD44⁺CD122⁺Helios⁺ CD8⁺ Treg also express Kir, and CD44⁺CD122⁺Helios⁺ Kir⁺CD8⁺ Treg from the population can be administered to human subjects in an amount sufficient to ameliorate symptoms of autoimmune disease.

In some embodiments, the T cells isolated from the subject are enriched for CD8⁺ cells prior to growing the isolated cells in the culture medium. In some embodiments, CD8⁺ T cells are isolated prior to growing them in the culture medium. As used herein, the term “isolated” refers to removal of a sample comprising T cells from the subject and further separating the T cells from other non-T cells. In some embodiments, the cells are grown in RPMI medium supplemented with 10% FCS, β-mercaptoethanol, 1M Sodium Pyruvate, 10 mM Hepes Buffer, 2 mM L-Glutamine, 50 Units/ml Penicillin and 50 μg/ml Streptomycin. In some embodiments, the isolated cells are grown until the population of the specialized regulatory CD8⁺ T cells in the isolated cells increases in number by at least 5%, 10%, at least 20%, at least 30%, at least 50%, at least 75%, at least 90%, at least 100%, or at least 200%. In some embodiments, after the isolated T cells are grown, the specialized regulatory CD8⁺ T cells are further enriched by depleting one or more cell types other than the CD44⁺CD122⁺Helios⁺ CD8⁺ Treg cells, the CD44⁺CD122⁺Helios⁺Kir⁺ CD8⁺ Treg cells and the CD44⁺CD122⁺Kir⁺ CD8⁺ Treg cells. In some embodiments, the specialized regulatory CD8⁺ T cells are administered to the subject by intravenous injection. The specialized regulatory CD8⁺ T cells are separated from the culture medium before administration to the subject. The specialized regulatory CD8⁺ T cells are administered to the subject in an amount effective to ameliorate a symptom of the autoimmune disease.

In some embodiments, the specialized regulatory CD8⁺ T cells are administered in combination with an autoimmune drug. Non-limiting examples of such drugs include methotrexate, cyclophosphamide, Imuran (azathioprine), cyclosporin, and steroid compounds such as prednisone and methylprednisolone.

The specialized regulatory CD8⁺ T cells depend on IL-15 for activation of suppressive activity. Accordingly, aspects of the invention involve methods of treating autoimmune diseases by enhancing the activity of the specialized regulatory CD8⁺ T cells using an interleukin-15 receptor (IL-15R) agonist in an amount effective to ameliorate a symptom of the autoimmune disease.

In some embodiments, a subject in need of treatment of autoimmune disease is a subject identified as having an autoimmune disease, i.e. the subject has been diagnosed by a physician (e.g., using methods well known in the art) as having an autoimmune disease. In some embodiments, the subject in need of treatment is a subject suspected of having or developing an autoimmune disease, such as a subject presenting one or more symptoms indicative of an autoimmune disease. In some embodiments, a subject suspected of having an autoimmune disease may display abnormal titers of autoantibodies. The subject having abnormal titers of autoantibodies may have at least one other symptom of autoimmune disease or may be without other symptoms associated with autoimmune disease. The term “subject in need of treatment” further includes people who once had an autoimmune disease but whose symptoms have ameliorated.

The subject is an animal, typically a mammal. In one aspect, the subject is a dog, a cat, a horse, a sheep, a goat, a cow or a rodent. In important embodiments, the subject is a human. It is understood that humans and mice share similar T cell markers in many instances designated similarly. For example, human and mice both have CD44, CD122 and CD8. In case of one marker important to the subject matter of the invention, the designation is different. The marker Ly49 in mice is designated Kir (immunoglobulin-like killer inhibitory receptors) in humans. To the extent that the claims and description refer to the treatment of humans and markers and antibodies are discussed, it is intended that the marker is human and the antibody binds the human marker.

As used herein, a compound is an IL-15R agonist when, like IL-15, it binds IL-15R and induces proliferation/activation of the specialized regulatory CD8⁺ T cells. In some embodiments, the agonist induces a biological response that is of a similar or higher level than the one induced by native IL-15. Some agonists induce an even higher level of biological response (super-agonist). An IL-15R agonist typically has an affinity for binding to IL-15 receptor alpha and/or to IL-15 receptor beta/gamma that is similar to that of native IL-15. Examples of such agonists include, but are not limited to: IL-15 polypeptide having the amino acid sequence of SEQ ID NO:1 (IL-15) and polypeptides that comprises a sequence at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO:1 (IL-15); agonistic anti-IL-15R antibody, or an antigen-binding fragment thereof; heterocomplex of IL-15 linked to soluble IL-15 receptor alpha chain (sushi domain) via a GS linker (recombinant IL-15/IL-15RA complex) (see e.g., U.S. application Ser. No. 12/090,930 the entire contents of which are incorporated by reference). In any of the embodiments, the IL-15R may be human IL-15R and the IL-15R receptor agonist is an agonist of human IL-15R.

The amino acid sequence of human IL-15 is represented by NCBI Reference Sequence: NP_(—)000576.1 (SEQ ID NO:1). IL-15 polypeptides also include fragments of IL-15, such as amino acids 49-162 of SEQ ID NO:1, which has previously been characterized as a mature form of IL-15 derived by proteolytic cleavage of a leader sequence from the polypeptide of SEQ ID NO:1, and other fragments that retain the biological activity of IL-15 are encompassed by the term IL-15. IL-15 analogs, including derivative or variants of IL-15 having one or more substituted amino acid, that exhibit the biological activity of IL-15 are also included within the meaning of the term IL-15R agonist. Exemplary analogs are described in U.S. application Ser. No. 11/639,877, U.S. Pat. No. 5,552,303, and in Bernard et al, J. Biol. Chem. 279:24313-24322, 2004, which are incorporated herein by reference. The term, IL-15 also encompasses IL-15 of species other than human, such as nonhuman primates, mouse, rat, pig, horse, cow, dog, etc.

A self antigen (or auto-antigen) is a subject's self-produced constituent, against which the subject mounts an undesired immune response. An “autoantibody” is an antibody produced by a subject, which binds to one or more of the subject's own constituents or self antigens. The term ‘autoimmune disease’ refers to those disease states and conditions wherein the immune response of the patient is directed against the patient's own constituents resulting in an undesirable and often terribly debilitating condition. As used herein, ‘autoimmune disease’ is intended to further include autoimmune conditions, syndromes and the like. Example of autoimmune diseases include, but are not limited to systemic lupus erythematosus, chronic graft versus host disease, rheumatoid arthritis, insulin-dependent diabetes mellitus, multiple sclerosis, psoriasis, inflammatory bowel disease, Sjogren's syndrome, Graves disease, Crohn's disease, Waldenstrom's macroglobulinemia, hyperviscosity syndrome, monoclonal gammopathy of undetermined origin, POEMS syndrome, myeloma, macroglobulinemia, and cold agglutinin disease. In some embodiments, the autoimmune disease involves antibodies to a self antigen and the subject has the antibodies to the self antigen.

The specialized regulatory CD8⁺ T cells or the IL-15R agonist are administered in an effective amount. An effective amount is a dose sufficient to provide a medically desirable result and can be determined by one of skill in the art using routine methods. In some embodiments, an effective amount is an amount which results in any improvement in the condition being treated. In some embodiments, an effective amount may depend on the type and extent of the autoimmune disease or condition being treated and/or use of one or more additional therapeutic agents. However, one of skill in the art can determine appropriate doses and ranges of therapeutic agents to use, for example based on in vitro and/or in vivo testing and/or other knowledge of compound dosages.

When administered to a subject, effective amounts of the therapeutic agent will depend, of course, on the particular disease being treated; the severity of the disease; individual patient parameters including age, physical condition, size and weight, concurrent treatment, frequency of treatment, and the mode of administration. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. In some embodiments, a maximum dose is used, that is, the highest safe dose according to sound medical judgment.

In the treatment of autoimmune disease, an effective amount is that amount which slows the progression of the disease, halts the progression of the disease, or reverses the progression of the disease. An effective amount includes that amount necessary to slow, reduce, inhibit, ameliorate or reverse one or more symptoms associated with the autoimmune disease. In some embodiments, such terms refer to a reduction in the swelling of one or more joints or a reduction in the pain, fatigue and/or fever associated with an autoimmune disorder. In some embodiments, such terms refer to a reduction in the levels of circulating autoantibodies associated with the autoimmune disease. In some embodiments, such terms refer to a reduction in a human's PASI score. In some embodiments, such terms refer to an improvement in a human's global assessment score.

An effective amount of a compound typically will vary from about 0.001 mg/kg to about 1000 mg/kg in one or more dose administrations, for one or several days (depending of course of the mode of administration and the factors discussed above).

Actual dosage levels of the therapeutic agent can be varied to obtain an amount that is effective to achieve the desired therapeutic response for a particular patient, compositions, and mode of administration. The selected dosage level depends upon the activity of the particular compound, the route of administration, the tissue being treated, and prior medical history of the patient being treated. However, it is within the skill of the art to start doses of the compound at levels lower than required to achieve the desired therapeutic effort and to gradually increase the dosage until the desired effect is achieved.

According to some aspects of the invention, a composition is provided. The composition comprises CD44⁺CD122⁺Kir⁺ CD8⁺ Treg cells wherein at least 5% of the CD8⁺ cells in the composition are CD44⁺CD122⁺Kir⁺ CD8⁺ Treg cells. In some embodiments, at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, at least 90%, at least 95% or 100% of the CD8⁺ cells in the composition are CD44⁺CD122⁺Kir⁺ CD8⁺ Treg cells. According to some aspects of the invention, a pharmaceutical composition comprising a composition of CD8⁺ cells, wherein at least 5% of the CD8⁺ cells in the composition are CD44⁺CD122⁺Kir⁺ CD8⁺ Treg cells, and a pharmaceutically acceptable carrier is provided. In some embodiments, the pharmaceutical composition comprises an immunosuppressive agent. Examples of immunosuppressive agents include, but are not limited to, non-steroidal anti-inflammatory agents, cyclosporine A, FK506, anti-CD4 antibody and rapamycin.

Some aspects of the invention involve a method for stimulating an immune response to an antigen. The method comprises administering to a subject having the antigen and in need of immune stimulation to the antigen an agent that inhibits the proliferation and/or activity of CD44⁺CD122⁺Kir⁺ CD8⁺ Treg cells in an amount effective to stimulate an immune response in the subject to the antigen.

In some embodiments, the subject has an infection with a pathogen and the pathogen comprises the antigen. As used herein, “infection” refers to invasion of the body of a subject with an organism that has the potential to cause disease. Such organisms include, but are not limited to, viruses, bacteria, fungi and parasites.

Examples of viruses include, but are not limited to, Human immunodeficiency viruses (HIV-1 and HIV-2), Human T lymphotrophic virus type I (HTLV-I), Human T lymphotrophic virus type II (HTLV-II), Herpes simplex virus type I (HSV-1), Herpes simplex virus type 2 (HSV-2), Human papilloma virus (multiple types), Hepatitis A virus, Hepatitis B virus, Hepatitis C and D viruses, Epstein-Ban virus (EBV), Cytomegalovirus and Molluscum contagiosum virus.

Examples of bacteria include, but are not limited to, Pasteurella species, Staphylococci species, Streptococcus species, Escherichia coli, Pseudomonas species, and Salmonella species. Specific examples of infectious bacteria include but are not limited to, Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia, Mycobacteria sps (e.g. M. tuberculosis, M. avium, M. intracellular, M. kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus {anaerobic sps.), Streptococcus pneumoniae, pathogenic Campylobacter sp., Enterococcus sp., Haemophilus influenzae, Bacillus antracis, corynebacterium diphtheriae, corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema palladium, Treponema pertenue, Leptospira, Rickettsia, and Actinomyces israelii.

Examples of fungi, but are not limited to, include Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, Candida albicans. Other infectious organisms (i.e., protists) include Plasmodium spp. such as Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, and Plasmodium vivax and Toxoplasma gondii. Blood-borne and/or tissues parasites include Plasmodium spp., Babesia microti, Babesia divergens, Leishmania tropica, Leishmania spp., Leishmania braziliensis, Leishmania donovani, Trypanosoma gambiense and Trypanosoma rhodesiense (African sleeping sickness), Trypanosoma cruzi (Chagas' disease), and Toxoplasma gondii.

Examples of parasites include, but are not limited to, Plasmodium spp., Babesia microti, Babesia divergens, Leishmania tropica, Leishmania spp., Leishmania braziliensis, Leishmania donovani, Trypanosoma gambiense and 60 Trypanosoma rhodesiense (African sleeping sickness), Trypanosoma cruzi (Chagas' disease), and Toxoplasma gondii.

In some embodiments, the subject has cancer, the cancer comprises the antigen, and the antigen is a tumor antigen. The cancer can be any cancer. Cancers include, but are not limited to, biliary tract cancer; bone cancer; brain and CNS cancer; breast cancer; cervical cancer; choriocarcinoma; colon cancer; connective tissue cancer; endometrial cancer; esophageal cancer; eye cancer; gastric cancer; Hodgkin's lymphoma; intraepithelial neoplasms; larynx cancer; lymphomas; liver cancer; lung cancer; melanoma; neuroblastomas; oral cavity cancer; ovarian cancer; pancreas cancer; prostate cancer; rectal cancer; sarcomas; skin cancer; testicular cancer; thyroid cancer; and renal cancer.

The tumor antigen can be any tumor antigen. For example, the tumor antigen can be, but is not limited to human epithelial cell mucin (Muc-1; a 20 amino acid core repeat for Muc-1 glycoprotein, present on breast cancer cells and pancreatic cancer cells), the Ha-ras oncogene product, p53, carcino-embryonic antigen (CEA), the raf oncogene product, GD2, GD3, GM2, TF, sTn, MAGE-1, MAGE-3, tyrosinase, gp75, Melan-A/Mart1, gplOO, HER2/neu, EBV-LMP 1 & 2, HPV-F4, 6, 7, prostatic serum antigen (PSA), Prostate Specific Membrane Antigen (PSMA), alpha-fetoprotein (AFP), CO17-1A, GA733, gp72, p53, the ras oncogene product, HPV E7 and melanoma gangliosides. In some embodiments, the antigen is administered to the subject in a vaccine. This administration may supplement the presence of the antigen in the subject due to an infection or the existence of a tumor. The vaccine may also involve the introduction into the subject of the antigen for the first time. As used herein, “vaccine” means an organism or material that contains an antigen in an innocuous form. The vaccine may be recombinant or non-recombinant. Vaccines may take the form, for example, of a toxoid, which is defined as a toxin that has been detoxified but that still retains its major immunogenic determinants; or a killed organism, such as typhoid, cholera and poliomyelitis; or attenuated organisms that are the live but non-virulent, forms of pathogens, or it may be antigen encoded by such organism, or it may be a killed tumor cell or an antigen present on a tumor cell.

Antigens include but are not limited to cells, cell extracts, proteins, polypeptides, peptides, polysaccharides, polysaccharide conjugates, peptide and non-peptide mimics of polysaccharides and other molecules, small molecules, lipids, glycolipids, carbohydrates, viruses and viral extracts and multicellular organisms such as parasites and allergens (see e.g., U.S. Pat. No. 7,576,066).

An agent that inhibits the proliferation and/or activity of specialized regulatory CD8⁺ T cells includes, but is not limited to, anti IL-15 antibody or an antibody that binds the specialized regulatory CD8⁺ T cells. The agent can be an anti-CD8 antibody or an antibody to Kir. In some embodiments, the antibody that binds CD44⁺CD122⁺Kir⁺ CD8⁺ Treg cells is a bispecific antibody comprising a first antigen-binding domain that binds to a first surface marker present on the CD44⁺CD122⁺Kir⁺ CD8⁺ Treg cells and a second antigen-binding domain that binds to a second surface marker present on the CD44⁺CD122⁺Kir⁺ CD8⁺ Treg cells. The first and second surface markers on the CD44⁺CD122⁺Kir⁺ CD8⁺ Treg cells include, but are not limited to, CD44, CD 122, Kir, and CD8. In some embodiments, the agent is a composition comprising a sub-optimal dose of a first antibody that binds a first surface antigen present on CD44⁺CD122⁺Kir⁺ CD8⁺ Treg cells, and a sub-optimal dose of a second antibody that binds a second surface antigen present on the CD44⁺CD122⁺Kir⁺ CD8⁺ Treg cells, wherein the first surface antigen is different from the second surface antigen. The first antibody binds an antigen selected from the group consisting of CD44, CD122, Kir and CD8 and the second antibody binds an antigen selected from the group consisting of CD44, CD 122, Kir, and CD8.

In some embodiments, the agent that inhibits the proliferation and/or activity of specialized regulatory CD8⁺ T cells is an agent that decreases the expression levels and/or activity of Helios in the cells. The agent that inhibits Helios levels and/or activity can be one or more of: a Helios binding protein that binds and inhibits Helios activity, e.g., DNA binding activity, nuclear translocation activity, homo or heterodimerization activity, or transcriptional activation activity; an antibody that specifically binds to the Helios protein, e.g., an antibody that disrupts Helios' ability to bind DNA or another transcription factor, to translocate to the nucleus, or bind DNA; a mutated inactive Helios or fragment thereof which, e.g., binds to Helios binding partner (e.g., DNA or another transcription factor, e.g., Ikaros or Aiolos factor) but disrupts Helios activity, e.g., nuclear translocation activity or transcriptional activation activity; a Helios nucleic acid molecule that can bind to a cellular Helios nucleic acid sequence, e.g., mRNA, and inhibit expression of the protein, e.g., an antisense molecule, siRNA; an agent which decreases Helios gene expression, e.g., a small molecule which binds the promoter of Helios and decreases Helios gene expression.

In some embodiments, the agent is a an agent that recognizes one of the markers present on the specialized regulatory CD8⁺ T cells coupled to a toxin. The agent that recognizes one of the markers present on the CD44⁺CD122⁺Kir⁺ CD8⁺ Treg cells could be a ligand specific to one of the markers, or an antibody to one of the markers. Examples of toxins include, but are not limited to biological toxins, chemical toxins, synthetic toxins and radionuclides. The radionuclides can be selected from the group consisting of beta emitting metallic radionuclides, alpha emitters and gamma emitters. Examples of radionuclides include, but are not limited to, ¹²³I, ¹²⁵I, ¹³⁰I, ¹³¹I, ¹³³I, ¹³⁵I, ¹²⁸Ba, ¹³N, ¹⁵O, and ¹⁸F.

As used herein, a sub-optimal amount of an antibody is an amount that if used alone would not be an effective and/or optimal amount to inhibit the activity of the specialized regulatory CD8⁺ T cells to treat the condition, but when used in combination with the second antibody would be an effective and/or optimal amount to inhibit the activity of the specialized regulatory CD8⁺ T cells to treat the condition. The use of sub-optimal doses of therapeutic agents is useful because it allows for a reduction in any potential side effects of the antibodies.

The agent that inhibits the proliferation and/or activity of the specialized regulatory CD8⁺ T cells is administered in an amount effective to stimulate an immune response to the antigen in the subject. The term “effective amount” as provided herein, refers to a sufficient amount of the agent to provide an immunological response and corresponding therapeutic effect. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, and the particular agent, mode of administration, and the like. An appropriate “effective” amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.

Pharmaceutical preparations and compounds are administered to a subject by any suitable route. For example, compositions can be administered orally, including sublingually, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically and transdermally (as by powders, ointments, or drops), bucally, or nasally. The pharmaceutical preparations of the present invention may include or be diluted into a pharmaceutically-acceptable carrier. The term “pharmaceutically-acceptable carrier” as used herein means one or more compatible fillers, diluants or other such substances, which are suitable for administration to a human or other mammal such as a dog, cat, or horse. The term “carrier” denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The carriers are capable of being commingled with the preparations of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy or stability. Carriers suitable for oral, subcutaneous, intravenous, intramuscular, etc. formulations can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa.

The present invention is further illustrated by the following Example, which in no way should be construed as further limiting. The entire contents of all of the references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference.

EXAMPLE Materials and Methods Mice.

C57BL/6J (B6), Rag2^(−/−), IL-15^(−/−) and B6.Qa-1(D227K) mice (backcrossed for 11 generations) were housed in pathogen-free conditions. All experiments were performed in compliance with federal laws and institutional guidelines as approved by Dana Farber Cancer Institute's Animal Care and Use Committee.

Reagents and Flow Cytometry.

Single-cell suspensions were prepared and maintained in the dark at 4° C. for immunofluorescent analysis, washed in ice-cold FACS buffer (2% fetal calf serum, 0.1% NaN3 in PBS) and incubated with each antibody for 30 min and washed with FACS buffer before analyzing. Anti-CD4, anti-CD8, anti-B220, anti-CD44, anti-CD62L, anti-Fas, anti-ICOS, anti-IgM, anti-CD200 (BD Bioscience) or anti-CD8β, anti-Ly49C/I/F/H, anti-Ly49A, Ly-49G2, Ly49C/I, Ly49D, Ly49F, Ly49H (eBioscience) were used followed by analysis of cells using a FACSCanto (BD Biosciences) and FlowJo software (TriStar).

Cell Purification and Adoptive Transfer.

Naïve B cells were isolated from spleen of Qa-1 WT and mutant mice (BD Bioscience). Naïve CD4⁺CD25⁻ cells were purified from spleens of Qa-1 WT and mutant mice using a CD4 cell enrichment kit (BD Bioscience) and biotinylated anti-CD25 Ab. B cell and CD4 cell purity was >95%. To generate immune CD8 cells, WT B6 mice were immunized i.p. with 100 μg KLH in CFA and splenic CD8 cells obtained 7-10 days later (BD Bioscience) and sorted for CD44⁺CD122⁺Ly49⁺ CD8 T cells. These cells were transferred into Rag2^(−/−) recipients intravenously. Immediately after cell transfer, Rag2^(−/−) mice were immunized i.p. with 100 μg NP₁₉-KLH in CFA and reimmunization i.p. with 50 μg NP₁₉-KLH in IFA.

ELISA.

For detection of NP specific antibodies, ELISA plates were coated with 0.5 μg/ml NP₄-BSA or 1 μg/ml NP₂₃-BSA (Biosearch Technologies) and serum harvested 14 days after immunization with NP₁₉-KLH in CFA and reimmunization with NP₁₉-KLH in IFA was used as a standard. 1:4000 dilution of this immune serum was defined as 100 units/ml. Total IgG, IgG1 and IgG2a were detected by incubating plates with biotinylated anti-mouse IgG, IgG1 or IgG2a followed by streptavidin-peroxidase. For the detection of autoantibodies, porcine thyroglobulin (Sigma) and porcine insulin (Sigma) were used to detect relevant Ab.

Statistics.

Statistical analyses were performed using Wilcoxon-Mann-Whitney rank sum test for comparison of two conditions and Kruskal-Wallis test for comparison of more than two conditions. P value<0.05 was considered statistically significant (*=<0.05, **=<0.01, ***=<0.001).

Results CD8⁺ Treg Depend on IL-15 for Acquisition of Suppressive Activity

CD44⁺ CD8⁺ cells are composed of a CD122⁺ IL-15-dependent fraction and a CD122⁻ IL-15-independent fraction (FIG. 1A). Analysis of CD8⁺ CD44⁺CD122⁺ cells from WT and IL-15^(−/−) mice showed that WT mice displayed 5-fold more CD8⁺ CD44⁺CD122⁺ cells compared to IL-15^(−/−) mice. Immunization of WT mice with KLH in CFA induced a ˜50% increase of CD44⁺CD122⁺ CD8 T cells but no detectable increase in IL-15^(−/−) mice (FIG. 1). Further analysis of the residual population of CD44⁺CD122⁺ CD8 cells from IL-15^(−/−) mice showed that these cells expressed considerably lower levels of CD122 (FIG. 1A, left panel).

These data suggest that defective suppressive activity of CD44⁺ CD8⁺ T cells from IL-15^(−/−) donors might reflect a failure to generate CD122⁺CD44⁺ CD8 T cells. To test Qa-1-dependent suppressive activity of CD44⁺CD122⁺ CD8 cells, we sorted CD44⁺CD122⁺ CD8⁺ cells (>98% purity) or CD44⁺CD122⁻ CD8⁺ cells (>96% purity) from WT B6 mice and transferred them into Rag2^(−/−) hosts along with (CD25⁻) CD4 cells from Qa-1 (WT) or Qa-1 (D227K) donors and B cells. After challenge with NP₁₉-KLH, recipients of CD44⁺CD122⁺ CD8 cells, but not CD44⁺CD122⁻ cells, displayed Qa-1-restricted suppression of NP-specific Ab responses (FIG. 1B). In contrast, the CD44⁺CD122⁻ CD8 cells did not suppress.

The generation of a high affinity Ab response is a precisely orchestrated process that depends on the activation of germinal center (GC) B cells by T_(FH) cells. The D227K mutation of Qa-1 disrupts the inhibitory interaction between CD8⁺ Treg and Qa-1⁺ target T_(H) cells. This defective inhibitory interaction allows robust autoantibody responses to thyroglobulin and insulin, even in the presence of high numbers of CD8⁺ Treg (FIG. 1C).

Expression of Ly49 by CD8⁺ Treg.

CD44⁺CD122⁺ CD8 T cells are divisible into two subsets according to expression of Ly49. The Ly49⁺ CD8 cells are particularly responsive to activation by IL-15 in vitro and depend on CD4⁺ T cells for efficient development and age-dependent expansion in vivo (Coles et al., 2000; Anfossi et al., 2004).

Immunization of WT B6 mice with KLH/CFA doubled the numbers of CD44⁺CD122⁺ CD8 T cells and ˜25% of these cells also express Ly49. These CD44⁺CD122⁺Ly49⁺ cells also express CD8αβ⁺ and not CD8αα as well as high levels of CD62L (FIG. 2 a). Additional analysis revealed that the Ly49⁺ CD8 subset expressed the following inhibitory receptors: Ly49A (24.8%), Ly49C/I (13.1%), Ly49F (90%) and Ly49G2 (33%); activating Ly49 receptors (Ly49D,H) were not expressed at detectable levels by these cells (FIG. 2 a).

Sorted CD44⁺CD122⁺Ly49⁺, but not CD44⁺CD122⁺Ly49⁻, CD8 T cells efficiently suppressed Qa-1 WT CD4 T cells, but not Qa-1 mutant (D227K) CD4 T cells, i.e. suppressive activity depended on recognition of Qa-1 on target CD4 cells (FIG. 2 b). These findings indicate that Ly49 expression can distinguish CD8⁺ Treg from conventional CD8⁺ T memory cells and open the possibility that development of CD8⁺ Treg may depend in part on engagement of Ly49 by class I MHC.

Defective CD8 Treg Function in B6-Yaa May Contribute to the Development of Lupus-Like Disease Expression of a point mutation that disrupts the interaction of Qa-1 ligands with the CD8/TCR complex (Lu et al., 2008) results in the development of a lupus-like autoimmune disorder in B6.Qa-1(D227K) mice associated with dysregulated expansion of T_(FH) (Kim et al., 2010). Defective CD8⁺ Treg activity may also contribute to disease pathogenesis in BXSB-Yaa mice based on findings that a cross between BXSB-Yaa mice and B6.β₂m^(−/−) mice shows accelerated onset and intensity of lupus-like disease (Bubier et al., 2007). The autoimmune disorder of BXSB-Yaa males, mainly attributed to a Y chromosome locus derived from the SB/Le strain, in fact represents an extra translocated copy of TLR7 onto the Y chromosome. This Y-linked extra copy of TLR7 exacerbates disease in a number of lupus-prone strains (Izui et al., 1988; Morel et al., 2000; Pisitkun et al., 2006) and promotes disease in non-autoimmune C57BL/6 background (B6-Yaa). Preliminary analysis of B6-Yaa mice showed that both 2 and 8 mo old mice harbored an approximate three-fold increase of T_(FH) and GC B cells in spleen, reminiscent of the B6.Qa-1(D227K) phenotype (FIG. 3). We asked whether the autoimmune phenotype in B6-Yaa mice was associated with defective regulation by CD8⁺ Treg. The numbers of CD44⁺CD122⁺Ly49⁺ CD8⁺ T cells in B6-Yaa mice were twice the number found in B6 WT mice 7 days after KLH/CFA immunization (data not shown). However, transfer of CD44⁺CD122⁺ CD8⁺ Treg from B6-Yaa mice along with B6 (WT) CD4 cells and B cells into Rag2^(−/−) hosts revealed that B6-Yaa CD8 Treg were unable to suppress target CD4 T cells. Since CD8 cells from WT mice transferred robust suppression in the same experiment (FIG. 4), this finding opens the possibility that T_(FH) cell expansion associated with autoimmunity in B6-Yaa mice reflects defective CD8 Treg function. Vaccination with Suboptimal Levels of Irradiated B16-GM-CSF-Transduced Cells Confers Increased Survival after B16 Tumor Inoculation in Mice with Defective CD8⁺ Treg Activity

C57BL/6 Qa-1 (D227K) knock in mice and wild type C57BL/6 mice were vaccinated in one flank subcutaneously with 2×10⁵ with GM-CSF-irradiated (3500 rads) B16 cells at time 0. One week after the vaccination, the mice were inoculated subcutaneously with 5×10⁶ B16 melanoma cells in the opposite flank (Dranoff et al, Proc Natl Acad Sci USA. 1993 Apr. 15; 90(8):3539-43; Sampson et al, Proc Natl Acad Sci USA. 1996 Sep. 17; 93(19):10399-404). The C57BL/6 Qa-1 (D227K) knock in mice with defective CD8⁺ Treg activity survived longer than the wild type C57BL/6 mice (FIG. 7).

A subpopulation of CD8⁺ T cells programmed to regulate CD4⁺ follicular helper T cell (TFH) activity through recognition of Qa-1 expressed at the surface of TFH cells dampens autoantibody responses (Kim et al., 2010; the entire content of which is incorporated by reference herein). Previous analysis of the surface phenotype of these CD8⁺ regulatory (Treg) cells indicated that they expressed high levels of CD44 and CD122 and relatively lower levels of CXCR5 and ICOSL. Expression of CD122 on their surface is in accord with their dependence on IL-15 for both development and function (Kim et al., 2010). The regulatory activity is invested in the subpopulation of CD44⁺CD122⁺ CD8⁺ cells that also express the Kir receptor. Expression of the class I MHC receptor by Ly49 by CD8 cells has been associated with enhanced responsiveness to IL-15 (Coles et al., 2000; Judge et al., 2002; Anfossi et al., 2004). These findings indicated that CD44⁺CD122⁺Kir⁺ CD8 T cells, which represent 3-5% of CD8 T cells, account for virtually all of the Qa-1-restricted suppressive activity invested in this T cell subset.

Analysis of Qa-1 knock-in mice that express an amino acid exchange mutation (D→K) at position 227, which disrupts the interaction of Qa-1 ligands with the CD8/TCR complex, revealed defective CD8⁺ Treg activity that was associated with expansion of TFH and GC B cells. These cellular abnormalities were associated with development of a lupus-like autoimmune disorder characterized by tissue-specific autoantibodies, lymphocyte infiltration of non-lymphoid organs and severe glomerulonephritis, a constellation of pathological changes similar to the syndrome displayed by BXSB-Yaa and C57BL/6-Yaa mice. The finding that BXSB-Yaa disease is exacerbated by the absence of β2m-associated class Ia/Ib proteins (Kim et al., 2010) also is consistent with the hypothesis that defective CD8⁺ Treg activity might contribute to BXSB-Yaa disease. We find that within the first several months life B6-Yaa mice contain increased numbers of TFH and GC B cells, and CD8⁺ Treg from these mice are unable to suppress WT CD4 T cells in adoptive hosts. Defects/abnormalities in the development of CD8⁺ Treg cells may contribute to disease pathogenesis in this murine model of lupus.

Regulatory CD8⁺ T cells are essential for maintenance of self-tolerance and inhibition of SLE-like autoimmune disease. The data presented herein establish the surface phenotype of CD8 Treg based on their dependence on the IL-15 cytokine for development and function. The results revealed that Qa-1-restricted suppressive activity of CD8 T cells is invested in a small subpopulation that expresses CD44, CD122 and Ly49 at their surface. In addition, we find that development of SLE-like disease in B6.Yaa autoimmune prone mice is associated with the defective regulatory activity of Qa-1-restricted CD8 Treg.

Qa-1, the mouse homolog of human leukocyte antigen E (HLA-E), forms a heterodimer with

₂-microglobulin that binds to and presents peptides derived from self or foreign proteins after deliberate immunization or infection (Lo et al., 1999; Lo et al., 2000; Sullivan et al., 2002). Although the mRNA encoding Qa-1 is detectable in many cell types (Transy et al., 1987), surface expression of the Qa-1-β₂-microglobulin heterodimer may be constrained by requirements for precise assembly and transport to the surface of activated T and B lymphocytes and dendritic cells (Sullivan et al., 2002; Soloski et al., 1995). Peptide-containing Qa-1 complexes engage two broad classes of receptors. Qa-1 heterodimers containing peptides derived from MHC class Ia leader sequences, called Qdm (for Qa-1 determinant modifier), bind to nonclonally distributed CD94-NKG2A receptors expressed by natural killer (NK) cells and a subpopulation of CD8 T cells. The functional consequence of Qa-1/Qdm-NKG2A interactions is generally inhibition of NK or CD8 cytolytic activity (Moser et al., 2002). A second class of Qa-1 ligand comprises Qa-1-β₂-microglobulin heterodimers that contain a growing list of peptides (Tompkins et al., 1998; Lo et al., 2000; Jiang et al., 2003; Davies et al., 2003). Interaction between this set of Qa-1 ligands and the TCR on CD8 T cells can promote CD8⁺ Treg induction, population expansion and expression of effector cell activity.

Recent analysis of Qa-1 mutant mice has defined the essential contribution of CD8⁺ Treg in the inhibition of autoimmune disease: the preferential target of CD8⁺ Treg is the T_(FH) subset, which expresses high steady-state levels of Qa-1 at their surface. Genetic disruption of the inhibitory interaction between CD8 Treg and target Qa-1⁺ follicular helper T cells resulted in the development of a SLE-like autoimmune disease that was facilitated by stimulation with protein or viral antigens (Kim et al., 2010). Perforin and IL-15 have been shown to be required for the complete activity of CD8 Treg. An IL-15 dependent trans cytotoxic mechanism has been shown to be efficiently enhanced by IL-21, the canonical cytokine expressed by activated T_(FH) cells (Zeng et al., 2005).

Our analysis revealed that CD8⁺ Treg express inhibitory Ly49 receptors (Ly49A, Ly49G2, Ly49C/I, Ly49F), especially Ly49F (FIG. 2A), but not activating receptors. Earlier studies have shown that CD8 T cells expressing inhibitory Ly49 family members display a CD4-dependent increase with age, while STAT1-deficiency reduces the size of this Ly49⁺ CD8 subpopulation (Coles et al., 2000; Anfossi et al., 2004). Although engagement of Ly49 can inhibit CD8 T cell activation (Coles et al., 2000), expression of inhibitory Ly49 molecules may also decrease activation induced cell death (AICD) and thus protect this memory population from premature elimination (Ugolini et al., 2001; Young et al., 2001; Roger et al., 2001; Chwae et al., 2002; Gati et al., 2003). These considerations suggest that inhibitory expression of Ly49 receptor on the surface of CD8⁺ Treg may limit levels of initial activation but prolong their lifespan in the memory pool.

Interestingly, the ligand(s) that bind to the Ly49F receptor, the major Ly49 family member expressed by CD8⁺ Treg, has not been identified. Lack of binding activity of Ly49F to conventional MHC class I molecules has led to speculation that this Ly49 family member may interact with non-classical MHC class 1 molecules (Kronenberg JI 2010). Human CD8 T cells also include a small subset of cells that expresses inhibitory KIR receptors, analogous to murine Ly49. Ly49 and KIR recognize mouse and human MHC class I, respectively. Ly49 proteins have a C-type lectin like domain in their extracellular domain, while the KIR family, as human NK inhibitory receptors, has an Immunoglobulin domain. Both proteins share structural homology with intracytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIM), which deliver inhibitory signals upon binding to MHC class I by their respective KIR or Ly49 receptors (Vivier and Anfossi, 2004). KIR⁺CD8⁺αβ⁻TCR⁺ T cells express a memory phenotype but generally do not express CCR7 and cell surface CD28/CD27 (Mingari et al., 1996; Speiser et al., 1999; Anfossi et al., 2001). Interestingly, intracellular expression of perforin is confined to KIR⁺ human CD8 T cells, representing ˜4-5% of the CD8⁺ T cell pool from normal adults (Anfossi et al., 2004). Previous studies have shown that human CD8⁺ T cell clones isolated from a small fraction of the KIR⁺CD8αβ⁺TCR⁺ T cell pool (˜4-5% of total CD8⁺ T cells) recognize HLA-E (human homolog of Qa-1b), consistent with our findings that Ly49⁺ CD8 T cells in mice exert Qa-1-dependent immune suppression (Pietra et al., 2001; Moretta et al., 2003; Mestas and Hughes, 2004).

We have noted that CD44⁺CD122⁺ cells express CXCR5 and ICOSL, according to bead-dependent enrichment (Kim et al., 2010) and FACS-dependent cell sorting (FIG. 5). This surface phenotype is of interest because of its potential contribution to suppressive activity of CD8⁺ Treg. CXCR5 expression may allow navigation of CD8⁺ Treg to the B/CD4 T cell follicles, while expression of ICOSL by activated emigrant CD8 Treg may enhance its interaction with ICOS⁺ T_(FH) cells within the follicular microenvironment. However, detection of these two surface proteins by available antibodies is relatively inefficient, while antibodies specific for CD44, CD122, and Ly49 are preferable for efficient isolation of CD8 Treg from heterogeneous cell populations. Sorting of CD8⁺ cells based on expression of the triad of CD44, CD122 and Ly49 will facilitate further analysis of the molecular mechanism of suppression and definition of the genetic signature of this regulatory lineage in B6 mice.

The Yaa genetic modifier, which increases severity of SLE in male mice, was first identified from a cross between C57BL/6 female and SB/Le male that produced the BXSB hybrid line. In addition to BXSB strain, the Yaa Y chromosome exacerbates disease in a number of lupus-prone strains (Kikuchi et al., 2005; Subramanian et al., 2006). Introgression of Yaa onto the non-autoimmune B6 background (B6-Yaa) revealed that Yaa could produce SLE-like disease at 8-12m of age characterized by lupus nephritis. Although end organ autoimmune disease does not develop in B6-Yaa mice until 8-12m, we find that expansion of T_(FH) and GC B cells is apparent by 2 m after birth (FIG. 5). Accumulation of T_(FH) and GC B cells at this early stage was associated with defective regulatory activity of CD8 T cells. Chronic activation of CD8⁺ memory cells by activated TLR7^(hi) DC may preferentially support development of cytokine-secreting CD8⁺ memory cells rather than CD8⁺ regulatory cells.

The IL-15 dependence of Qa-1-restricted CD8 Treg cells is reflected in co-expression of CD122 and Kir and that CD44⁺CD122⁺Ly49⁺ CD8 cells mediate Qa-1-dependent suppressive activity. Interruption of the interaction between CD8 Treg and target CD4 T cells results in autoantibody generation and SLE-like disease. These findings indicate that enhancement of CD8 Treg activity represents a new approach to treatment of autoimmune disease.

Expression of the Transcription Factor Helios Dictates the Phenotype and Suppressive Activity of Ly49⁺ CD8 Treg

DNA microarray with cDNA prepared from Ly49⁺ and Ly49⁻ CD8⁺ T cells identified the transcription factor Helios as a potential master transcription factor for genes that are important for the CD8 Treg development and function. Helios is one of the Ikaros family members of zinc finger regulator. Helios is highly expressed at early stage of T cell development and also expressed in a subset (˜70%) of FoxP3⁺CD4⁺ Treg cells; however, Helios expression is not dependent on FoxP3 expression. Mature B cells, dendritic cells and myeloid cells do not express Helios. Helios expression and function in CD8 T cell pool has not been reported. Our analysis shows that Helios is exclusively expressed by Ly49⁺ CD8 Treg cells within CD8 T cell pool and expression of this transcription factor in Ly49⁺ T cell is associated with their suppressive function (FIG. 8).

Phenotypic Characterization of Helios Expressing Ly49⁺ CD8 T Cells

Additional characterization of Helios⁺ Ly49⁺ murine CD8⁺ Treg is helpful for the future identification of KIR⁺ CD8⁺ Treg cells in human. As demonstrated in FIG. 9, Helios expressing Ly49⁺ CD8 cells express higher level of VLA-2 and VLA-4, integrins that are involved in the cell-cell interaction. In addition, development of Helios⁺Ly49⁺ CD8 T cells is IL-15 dependent, since IL-15 KO mice are devoid of Helios⁺Ly49⁺ CD8 cells. Analysis of a critical transcription factor, Helios, for CD8 Treg activity in mouse adds one more important factor for the identification of CD8⁺ Treg cells in human and for the understanding their genetic nature.

CD8 Treg Mediated Immunotherapy in Autoimmune Disease: CIA Model

Pathogenic CD4⁺ T cells associated with organ-specific autoimmune diseases can be effectively suppressed via recognition of Qa-1 expressed by these target CD4⁺ cells by CD8⁺ Treg. These autoimmune disease models include Type I diabetes (T1D), experimental autoimmune encephalomyelitis (EAE) and collagen-induced arthritis (CIA) in which conventional TH1- and/or TH17-type CD4 cells are known to play a pathogenic role. We have tested the efficacy of CD8 Treg in the prevention and treatment of autoimmune disease using collagen induced arthritis model. Development of disease can be delayed by infusion of Qa-1-restricted CD8⁺ Treg, but not by memory-phenotype CD8⁺ T cells.

Stimulation of CD8⁺ Treg by IL-15 in vitro results in robust expansion of these cells and maintenance of surface phenotype and functional activity. In FIG. 10, in vitro expanded CD8 Treg efficiently prevent the development of CIA.

CD8 Treg Mediated Immunotherapy in Cancer

Blockade of CD8⁺ Treg activity led to enhanced tumor immunity in B16 melanoma model: vaccination of mice with GM-CSF-producing B16 melanoma delayed the progression of tumor growth in Qa-1 D227K mice compared to rapid tumor growth in WT mice. Inhibition of tumor growth in Qa-1 D227K mice correlated with increased numbers of CD8⁺ effector T cell infiltration into tumors. Therefore, blockade of CD8⁺ Treg-dependent suppression will result in enhanced expansion and activity of effector T cells in tumor setting. To deplete CD8⁺ Treg, the expression of Ly49F subtype by more than 90% of Ly49⁺ CD8 Treg can be exploited. In fact, preliminary data has shown that depletion of Ly49F⁺ cells by anti-Ly49F antibody enhances anti-tumor immunity in B16 melanoma model. This finding suggests that administration of anti-KIR(X) antibody (antibody targeting the dominant KIR subtype in CD8 T cells in human) is a feasible intervention to enhance immune responses against cancer in human.

This invention is not limited in its application to the details of construction and the arrangement of components set forth in the above description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” or “having,” “containing”, “involving”, and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

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We claim:
 1. A method for treating an autoimmune disease comprising: administering to a subject in need of such treatment an interleukin-15 receptor (IL-15R) agonist in an amount effective to ameliorate a symptom of the autoimmune disease.
 2. The method of claim 1, wherein the autoimmune disease is selected from the group consisting of systemic lupus erythematosus, chronic graft versus host disease, rheumatoid arthritis, insulin-dependent diabetes mellitus, multiple sclerosis, psoriasis, inflammatory bowel disease, Sjogren's syndrome, Graves disease, Crohn's disease, Waldenstrom's macroglobulinemia, hyperviscosity syndrome, monoclonal gammopathy of undetermined origin, POEMS syndrome, myeloma, macroglobulinemia, and cold agglutinin disease.
 3. (canceled)
 4. The method of claim 1, wherein the agonist is a IL-15 polypeptide that comprises a sequence at least 90% identical to the amino acid sequence of SEQ ID NO:1 (IL-15) and binds an IL-15R.
 5. The method of claim 1, wherein the agonist is an anti-IL15R antibody or an antigen-binding fragment thereof.
 6. The method of claim 1, wherein the agonist is a heterocomplex of IL-15 linked to soluble IL-15 receptor alpha chain via a GS linker.
 7. A method for treating an autoimmune disease in a subject comprising: a. isolating T cells from the subject in need of such treatment, wherein the isolated T cells comprise a number of CD44+CD122+Helios+CD8+ Treg cells; b. growing the isolated cells in a culture medium containing IL-15 and IL-21 in the presence of Concavalin A activated and irradiated syngenic CD4 T cells until the number of CD44+CD122+Helios+CD8+ Treg cells increases by at least 5%, thereby producing a population of cells enriched with CD44+CD122+Helios+ CD8+ Treg cells; c. administering CD44+CD122+Helios+ CD8+ Treg cells from the population of cells to the subject in an amount effective to ameliorate a symptom of the autoimmune disease.
 8. The method of claim 7, wherein the CD44+CD122+Helios+ CD8+ Treg cells are Kir+. 9-12. (canceled)
 13. A method for treating an autoimmune disease in a subject comprising: a. isolating T cells from the subject in need of such treatment, wherein the isolated T cells comprise a number of CD44+CD122+Kir+CD8+ Treg cells; b. growing the isolated cells in a culture medium containing IL-15 and IL-21 in the presence of Concavalin A activated and irradiated syngenic CD4 T cells until the number of CD44+CD122+Kir+CD8+ Treg cells increases by at least 5%, thereby producing a population of cells enriched with CD44+CD122+Kir+ CD8+ Treg cells; c. administering CD44+CD122+Kir+ CD8+ Treg cells from the population of cells to the subject in an amount effective to ameliorate a symptom of the autoimmune disease. 14-17. (canceled)
 18. A composition comprising CD8+ cells, wherein at least 5% of the CD8+ cells in the composition are CD44+CD122+Helios+ CD8+ Treg cells.
 19. The composition of claim 18, wherein the CD44+CD122+Helios+ CD8+ Treg cells are Kir+.
 20. A composition comprising CD8+ cells, wherein at least 5% of the CD8+ cells in the composition are CD44+CD122+Kir+ CD8+ Treg cells. 21-24. (canceled)
 25. A method for stimulating an immune response to an antigen comprising: administering to a subject having the antigen and in need of immune stimulation to the antigen an agent that inhibits the proliferation and/or activity of CD44+CD122+CD8+ Treg cells in an amount effective to stimulate an immune response in the subject to the antigen, wherein the CD44+CD122+CD8+ Treg cells are Helios+ and/or Kir+. 26-27. (canceled)
 28. The method of claim 25, wherein the antigen is administered to the subject in a vaccine.
 29. The method of claim 25, wherein the agent is an anti IL-15 antibody.
 30. The method of claim 25, wherein the CD44+CD122+CD8+ Treg cells are Kir+ and the agent is an antibody that binds CD44+CD122+Kir+CD8+ Treg cells.
 31. The method of claim 25, wherein the CD44+CD122+CD8+ Treg cells are Helios+ and the agent inhibits the expression and/or activity of Helios.
 32. The method of claim 30, wherein and the antibody is a bispecific antibody comprising: a first antigen-binding domain that binds to a first surface marker present on the CD44+CD122+ Kir+CD8+ Treg cells; and a second antigen-binding domain that binds to a second surface marker present on the CD44+CD122+Kir+CD8+ Treg cells.
 33. The method of claim 32, wherein the first and second surface markers on the CD44+CD122+Kir+ CD8+ Treg cells are selected from the group consisting of CD44, CD122, Kir and CD8.
 34. (canceled)
 35. The method of claim 25, wherein the agent is a composition comprising a sub-optimal dose of a first antibody that binds a first surface antigen present on CD44+CD122+CD8+ Treg cells, and a sub-optimal dose of a second antibody that binds a second surface antigen present on the CD44+CD122+CD8+ Treg cells, wherein the first surface antigen is different from the second surface antigen.
 36. The method of claim 35, wherein the first antibody binds an antigen selected from the group consisting of CD44, CD122, Kir and CD8 and the second antibody binds an antigen selected from the group consisting of CD44, CD122, Kir and CD8.
 37. (canceled) 